Origin of the conformational heterogeneity of cardiolipin-bound cytochrome C.
Origin of the conformational heterogeneity of cardiolipin-bound cytochrome C.
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DOI：
10.1021/ja307426k
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发表时间：
2012-11-14
影响因子：
15
通讯作者：
Pletneva EV
中科院分区：
文献类型：
--
作者：
Hong Y;Muenzner J;Grimm SK;Pletneva EV
细胞色素c（cyt c）与心磷脂（CL）的相互作用部分地打开了激活其过氧化物酶功能的蛋白质，这是执行细胞凋亡的关键事件。然而，在异质系综中改变的蛋白质种类的结构特征难以用系综平均来探测。从时间分辨FRET（TR-FRET）的染料血红素距离分布P（r）的分析已经揭示了两种不同类型的CL结合的细胞色素c构象，扩展和紧凑。我们结合TR-FRET，荧光相关光谱（FCS），和生物层干涉法，以开发两种构象之间的功能划分的系统的理解。这两个亚群相互平衡，亚毫秒的构象交换率反映了蛋白质折叠成紧凑的非天然状态，以及蛋白质与脂质表面的相互作用。与带负电荷的脂质表面的静电相互作用与CL浓度的生理相关变化相关，强烈影响细胞色素C结合和构象交换的动力学。一个主要的外围结合机制，而不是深入的蛋白质插入到膜中，提供了一个基本原理的CL表面和大规模的蛋白质展开的一般变性效果。这些发现密切相关的细胞色素c折叠动力学，并建议延长时间窗口监测折叠动力学的一般策略。
Interactions of cytochrome c (cyt c) with cardiolipin (CL) partially unfold the protein activating its peroxidase function, a critical event in the execution of apoptosis. However, structural features of the altered protein species in the heterogeneous ensemble are difficult to probe with ensemble averaging. Analyses of the dye-to-heme distance distributions P(r) from time-resolved FRET (TR-FRET) have uncovered two distinct types of CL-bound cyt c conformations, extended and compact. We have combined TR-FRET, fluorescence correlation spectroscopy (FCS), and bio-layer interferometry to develop a systematic understanding of the functional partioning between the two conformations. The two subpopulations are in equilibrium with each other, with a sub-millisecond rate of conformational exchange reflecting the protein folding into a compact non-native state, as well as protein interactions with the lipid surface. Electrostatic interactions with the negatively-charged lipid surface that correlate with physiologically-relevant changes in CL concentrations strongly affect the kinetics of cyt c binding and conformational exchange. A predominantly peripheral binding mechanism, rather than deep protein insertion into the membrane, provides a rationale for the general denaturing effect of the CL surface and the large-scale protein unfolding. These findings closely relate to cyt c folding dynamics and suggest a general strategy for extending the time window in monitoring the kinetics of folding.
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